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acid catalysis
Industrial Crops & Products 128 (2019) 177–185
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Enhancing the solubility and antioxidant activity of high-molecular-weight lignin by moderate depolymerization via in situ ethanol/acid catalysis
T
Liangliang Ana, Chuanling Sia,b, , Guanhua Wanga,b,c, , Wenjie Suid, Zhengyi Taoa,b ⁎
⁎
a
Tianjin Key Laboratory of Pulp & Paper, College of Papermaking Science and Technology, Tianjin University of Science & Technology, Tianjin 300457, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China c Key Laboratory of Pulp and Paper Science & Technology of Ministry of Education of China, Qilu University of Technology, Jinan 250353, China d Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, China b
ARTICLE INFO
ABSTRACT
Keywords: Enzymatic hydrolysis lignin Mild depolymerization Antioxidant activity Solubility
Lignin, a by-product obtained from the pulping industry and cellulosic ethanol production, has received considerable attention for its antioxidant activity owing to its polyphenol structure. In this work, high-molecularweight lignin obtained by ethanol-water fractionation of enzymatic hydrolysis lignin, was moderately depolymerized via in situ ethanol/acid catalysis to improve its solubility and antioxidant activity. The depolymerized products showed significant reduction in molecular weight and improvement in solubility in an 80% ethanolwater (v/v) solution owing to the cleavage of ether linkages; this was confirmed by gel permeation chromatography (GPC), fourier transform-infrared spectroscopy (FT-IR), pyrolysis-gas chromatography/ mass spectrometer (Py-GC/MS), carbon-13 nuclear magnetic resonance spectrometry (13C NMR), and heteronculear single quantum coherence nuclear magnetic resonance spectrometry (HSQC NMR) analyses. In addition, the total phenolics content of the depolymerized products increased sharply, leading to a significant enhancement in the free radical scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl (DPPH). In particular, D-200 (depolymerized product at 200 °C) with IC50 = 66.0 ± 2.6 μg/mL was comparable to the positive control butylated hydroxytoluene (BHT) with IC50 = 53.3 ± 4.5 μg/mL, indicating the potential application of lignin as a high-valueadded antioxidant. In conclusion, the depolymerization of high-molecular-weight lignin via moderate ethanol/ acid catalysis may effectively improve its solubility and antioxidant activity.
1. Introduction Lignin, a heterogeneous natural polymer, consists of C6-C3 phenyl propane units including coniferyl alcohol (G), sinapyl alcohol (S), and p-coumaryl alcohol (H), and typically holds about 15–30% by weight and about 40% by energy in biomass (Doherty et al., 2011). Despite its enormous reserves and energy storage, lignin is underutilized in the current cellulose-dominant processing industries and is typically burned to generate power directly. Particularly with cellulosic ethanol production approaching commercial practices, immense quantities of enzymatic hydrolysis lignin are produced as a by-product. In addition, owing to the little use of other biomass components except for cellulose, the path of cellulosic ethanol is less economically competitive. Therefore, the high-value-added utilization of lignin plays a significant role in the commercial operation of lignocellulosic bio-refinery (Dai et al., 2017; Li et al., 2015).
As a natural and renewable compound with a polyphenol structure, lignin shows promising antioxidant activity, and several studies have focused on the exploration of its antioxidant properties (Tang et al., 2017; Wang et al., 2018c). García et al. evaluated the antioxidant activities of 14 lignin samples and discovered that autohydrolysis, organosolv, and alkali lignin exhibited similar antioxidant activities with natural antioxidant (García et al., 2012). With the advancement in research, the relationship between the structure and antioxidant capacity of lignin was further studied (Pan et al., 2006; Sun et al., 2014). It has been established that high molecular weight is a crucial factor that decreases the radical scavenging capacity of lignin (Aminzadeh et al., 2018). The incompatibility caused by poor solubility is also a vital issue that needs to be addressed (Laurichesse and Avérous, 2014). In our previous work, we demonstrated that lignin with low molecular weight and high total phenolics content exhibited preferable antioxidant activity (An et al., 2017). Therefore, obtaining low-molecular-weight
⁎ Corresponding authors at: Tianjin Key Laboratory of Pulp & Paper, College of Papermaking Science and Technology, Tianjin University of Science & Technology, Tianjin 300457, China. E-mail addresses: [email protected] (C. Si), [email protected] (G. Wang).
https://doi.org/10.1016/j.indcrop.2018.11.009 Received 10 April 2018; Received in revised form 13 September 2018; Accepted 3 November 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Extraction, fractionation, and depolymerization of enzymatic hydrolysis lignin (EHL).
lignin, increasing the total phenolics content, and improving the lignin solubility are considered feasible ways to improve its antioxidant activity. The selective acid-catalyzed depolymerization of lignin to obtain low-molecular-weight products has attracted immense interest in recent years. Lignin depolymerization by acid catalysis is a widely used method that breaks ether linkages to offer small segments including oligomeric and monomeric aromatic products (Li et al., 2015; Wang et al., 2018a). Although acid-catalyzed depolymerization of lignin in an aqueous system is a feasible technique to generate low-molecularweight products, the yield of the target products is often low owing to lignin condensation. Therefore, there have been attempts to inhibit condensation (Deuss et al., 2015; Güvenatam et al., 2014; Huang et al., 2015). For example, Ma et al. investigated the depolymerization of kraft lignin with multiple catalytic agents and noted that ethanol was an effective solvent in suppressing condensation and char-forming reactions (Ma et al., 2014). However, these studies mainly focused on identifying the promising substitutes of phenyl chemicals and liquid fuels owing to the growing shortage of fossil energy (Li et al., 2015), and less research has been performed on the relationship between lignin depolymerization and antioxidant activity. Thus, the hypothesis of this work is that the lignin antioxidant activity, which has a negative correlation with the molecular weight, may be enhanced by depolymerization owing to the decrease of molecular weight. In order to improve antioxidant activity, the high-molecularweight lignin part (insoluble in 80% ethanol) from the fractionation of enzymatic hydrolysis lignin was depolymerized in situ in 80% ethanol solvent via acid catalysis. Here, ethanol as a relatively cheap solvent from biomass bio-conversion, was used as the solvent for fractionation and depolymerization. The depolymerized products were characterized by gel permeation chromatography (GPC), fourier transform-infrared
spectroscopy (FT-IR), pyrolysis-gas chromatography/ mass spectrometer (Py-GC/MS), carbon-13 nuclear magnetic resonance spectrometry (13C NMR), and heteronculear single quantum coherence nuclear magnetic resonance spectrometry (HSQC NMR). The total phenolics content of the lignin samples was determined, and the free radical scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) was further measured to confirm the effect of the depolymerization on the antioxidant activity of lignin. 2. Material and methods 2.1. Materials Enzymatic hydrolysis residues (EHRs) of corn straw produced from steam explosion pretreatment and cellulosic enzymatic hydrolysis were kindly provided by Songyuan Laihe Chemical Co. Ltd, Jilin Province, China. All commercial chemicals were analytical reagents. 2.2. Extraction, fractionation, and depolymerization of lignin Enzymatic hydrolysis lignin (EHL) was isolated from EHRs by leaching in an aqueous solution of 1,4-dioxane (9:1, v/v), with a solid to liquid ratio of 1:10 (g/mL), assisted with a magnetic stirrer at room temperature for 12 h. After extraction, the mixture was centrifuged and the supernatant was concentrated by rotary evaporation to remove the solvent. After the addition of distilled water, the precipitate (EHL) was collected via centrifugation (Zikeli et al., 2016). Subsequently, 10 g EHL was added to 500 mL 80% ethanol-water solution, and the mixture was stirred with a magnetic stirrer at room temperature for 1 h followed by paper filtration (Wang and Chen, 2013a). The ethanol-insoluble lignin (EIL) was collected and then 178
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Table 1 Chemical composition of lignin samples: EHRs: enzymatic hydrolysis residues; EHL: enzymatic hydrolysis lignin. Lignin samples
Lignin/%
EHRs EHL
Carbohydrate/%
Acid-insoluble lignin
Acid-soluble lignin
Glucose
Xylose
Arabinose
52.20 ± 1.69 82.62 ± 1.05
2.38 ± 0.06 3.72 ± 0.12
20.55 ± 0.26 2.75 ± 0.69
6.88 ± 0.27 3.76 ± 0.19
0.73 ± 0.00 0.70 ± 0.06
depolymerized in situ in 80% ethanol. The depolymerization was conducted in a 150 mL stainless steel batch reactor. For this, 1.5 g EIL was suspended in 75 mL 80% ethanolwater solution (v/v, 4:1) that contained 0.10 mL 98% H2SO4, and the reaction mixture was heated to the desired reaction temperature under continuous stirring at 225 rpm for 1 h. After the reaction, the reactor was rapidly quenched with flowing water. The reaction mixture was filtered, and the filter cake (reaction residues) was dried to a constant weight. The filtrate was concentrated to a volume of 10 mL, followed by the addition of distilled water to precipitate product. The precipitant was washed to neutral pH with distilled water and collected as the depolymerized product. The flow-process diagram of extraction, fractionation, and depolymerization of lignin is shown in Fig. 1. The conversion rate of depolymerization was calculated by formula (1), and the yield of depolymerized products was calculated by formula (2):
conversion (%) =
Yield (%) =
mp mn
mn mr mn
× 100%
× 100%
100)/14.75*t )
11.25 ± 1.62 1.36 ± 0.00
dissolved in 0.5 mL dimethylsulfoxide-d6 (99.9%, Cambridge Isotopes Laboratories) to record the 13C NMR and HSQC NMR spectra (Sun et al., 2012). A single-shot pyrolyzer (Py-2020iS, Japan) coupled with a GC/MS (Agilent Technologies 5975C, America) i.e., a Py-GC/MS was employed to analyze the lignin structure according to literature (Dai et al., 2018; Liu et al., 2018). 2.4. Determination of the total phenolics content and antioxidant activity The total phenolics content of lignin was determined by the FolinCiocalteu (F-C) method (An et al., 2017). The DPPH free radical scavenging capacities for the lignin samples were evaluated in accordance with the protocol described in a previous literature (Lauberts et al., 2017).
(1)
3. Results and discussion 3.1. Lignin extraction, fractionation, and depolymerization
(2)
In order to minimize the destruction of the EHL structure during the extraction process, a mild method (using neutral 1,4-dioxane solvent at room temperature) was applied. After a quantitative comparison of lignin and polysaccharide content changes in the samples (Table 1), it was concluded that the mild extraction process was an effective way to provide a good substrate for the further depolymerization process. The total lignin content increased from 54.58% (EHRs) to 86.33% (EHL) and the increase in acid-insoluble lignin content was the dominant factor that elevated the purity of lignin. The content of acid-insoluble lignin in EHL increased to 82.62%, compared with 52.20% in EHRs. However, the content of acid-soluble lignin in EHL and EHRs did not show visible changes, which may be due to the assumption that the mild neutral solvent extraction process could effectively dissolve lignin and protect the chemical structure of lignin from destruction (Arni, 2018); thus, the acid-soluble lignin still occupied a small proportion of the whole lignin. In addition, the glucose content (2.75%) of EHL decreased sharply, and xylose and arabinose contents of EHL also decreased to 3.76% and 0.70%, respectively. Nevertheless, it was observed that the xylose content was higher than the content of glucose in EHL, which suggested that hemicellulose bonded covalently to lignin to form a lignin carbohydrate complex (LCC) was more difficult to remove during purification. The result agrees well with earlier studies (Lauberts et al., 2017; Wang and Chen, 2014). Next, the purified EHL was fractionated into two fractions by dissolution in 80% ethanol (shown in Fig. 1). The molecular weight distribution curves of EHL and EIL are presented in Fig. S1. From the chromatograms, the molecular weight curve of EIL apparently migrated to the high-molecular-weight section, while the low-molecular-weight section was distinctly weakened. The results indicated that the molecular weight of EIL was markedly higher than that of EHL. This corresponded well with the average molecular weight of EHL (M n = 5265 g/mol, M w = 11,595 g/mol, M w /M n = 2.20) and EIL (M n = 7731 g/mol, M w = 14,218 g/mol, M w /M n = 1.84). With a similar procedure, in our previous investigation, we successfully obtained alkaline-extracted lignin with high molecular weight from steam-exploded corn stalk through ethanol-water fractionation (Wang
where mn is the mass of ethanol-insoluble lignin for the depolymerization reaction in grams; mr is the mass of the reaction residues in grams; mp is the mass of depolymerized products in grams. The depolymerization effect was presented in relation to the “severity factor”, which combines the effects of treatment processing parameters (reaction temperature and reaction time) into a single equation. The severity factor, S0, for each treatment was calculated according to formula (3) (Martin-Sampedro et al., 2011).
S0 = log(e (T
Ash/%
(3)
where T is the reaction temperature in °C; t is the duration of the treatment in minutes. 2.3. Lignin characterization The lignin and carbohydrate contents of EHRs and EHL were determined using the two-step acid hydrolysis method (Sluiter et al., 2008; Wang and Chen, 2016). A modified GPC method using hydrophilic gel column (TSK G3000PWxl column) was employed to analyze the average molecular weight of the lignin samples according to literature (Wang and Chen, 2013b; Wang et al., 2018b). Lignin samples were dissolved in 1% NaOH and diluted with tris-acetate buffer (20 mmol/L, pH 7.4). The column was operated at 25 °C and eluted with tris-acetate buffer at a flow rate of 0.5 mL/min. subsequently, 20 μL samples were injected each time, and a UV-detector (280 nm) was used for detection. The molecular weight was calculated using the Agilent GPC data analysis system. FT-IR analysis was conducted by KBr pressing disc technique in the range of 4000-400 cm−1 using an FT-IR spectrophotometer (FT-IR-650, Gangdong Sci. & Tech. Co., Ltd, China) (Liu et al., 2017). 13 C NMR and HSQC NMR spectra were obtained using a Bruker AVANCE III 400 MHz spectrometer with a 5 mm high-resolution liquid probe. In this study, the lignin obtained via 1,4-dioxane-water solution extraction was readily dissolved in the DMSO-d6 solvent. According to the procedure, approximately 100 mg of the lignin sample was directly 179
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Table 2 Yield of depolymerized products under various temperatures *, and molecular weights of lignin.
* *
Run
Reaction temperature (°C)
S0*
Conversion rate of Depolymerization (%)
Yield of depolymerized products (%)
M n (g/mol)
M w (g/mol)
M w /M n (polydispersity index)
0 0 1 2 3
EHL* EIL* 160 180 200
– – 3.54 4.13 4.72
– – 94.29 94.89 96.69
– – 71.19 67.62 63.31
5265 7731 4742 3071 2856
11,595 14,218 8483 4906 4773
2.20 1.84 1.79 1.60 1.57
Reaction conditions: 80% ethanol-insoluble lignin 1.5 g, 80% ethanol solution 75 mL, 98% H2SO4 solution 0.1 mL, time = 1 h. EHL: enzymatic hydrolysis lignin. * EIL: ethanol-insoluble lignin. * S0: severity factor.
and Chen, 2013a). In order to improve the antioxidant activity of EIL, depolymerization by acid catalysis was performed in 80% ethanol solution (Fig. 1). Table 2 summarizes the yields of the depolymerized products (based on the average results of three parallel experiments). Here, the conversion rate of depolymerization represented the changes of lignin solubility in 80% ethanol solution. The severity factor (S0) increased from 3.54 (reaction temperature at 160 °C) to 4.13 (reaction temperature at 180 °C) and further to 4.72 (reaction temperature at 200 °C). When the reaction temperature was 160 °C, the conversion rate of depolymerization was 94.29%, which indicated that 94.29% of EIL was converted into a dissolvable form in 80% ethanol solution. Meanwhile, with the increase in S0, the conversion was also slightly enhanced, suggesting that the higher reaction temperature tended to promote the solubilization of lignin. However, with the increase in S0, the yield of the depolymerized products decreased. This phenomenon was possibly because the depolymerized lignin was further cracked down to lowermolecular-weight products that were hard to recover (Huang et al., 2015; Wang and Chen, 2013a). In summary, EIL may be depolymerized at a moderate temperature, and the depolymerized products were readily dissolved in 80% ethanol.
had a substantial effect on the molecular weight distribution of lignin. With the increase in S0, the molecular weight distribution curve further shifted towards the low-molecular-weight regions. At 200 °C, 96.69% of EIL was dissolvable in 80% ethanol-water solution, and the depolymerized product had the lowest molecular weight (M n = 2856 g/mol, M w = 4773 g/mol) and narrow polydispersity (M w /M n = 1.57) (Table 2). Compared to lignin depolymerization at higher temperatures (250–350 °C) in previous studies (Güvenatam et al., 2014; Ma et al., 2014), the reaction conditions in this work were comparatively moderate. In addition, as shown in the molecular weight distribution curves of the depolymerized products at different temperatures, lignin can be depolymerized effectively, and the depolymerized products were readily dissolvable in ethanol/water mixtures by moderate catalysis without the formation of agglomerates. This may be because ethanol could generate radicals as capping agents to eliminate the intermediate carbonium ions that are critical for lignin condensation (Huang et al., 2015; Voitl and Rudolf von Rohr, 2008). Hence, the GPC results of this work indicated that the moderate ethanol/acid catalysis can depolymerize high-molecular-weight lignin effectively and decrease the molecular weight significantly. 3.2.2. FT-IR analysis Nondestructive structural characterization by FT-IR was performed to interpret the chemical structure changes of lignin after depolymerization. The hydroxyl groups from phenolic and aliphatic groups at 3410 cm−1 appeared in all spectra (shown in Fig. 3) and a band centered at 2930 cm−1originating from the CeH-stretching in aromatic methoxyl groups, methyl, and the methylene group of side chains was also present (Rashid et al., 2018). In addition, aromatic skeleton vibrations were observed at 1600 cm−1, 1515 cm−1 and 1426 cm−1 in all lignin spectra. Peaks at 1260 cm−1 (guaiacyl unit, G), 1125 cm−1 (syringyl unit, S) and 834 cm−1 (CeH out-of-plane in S and H units)
3.2. Molecular weight and structural characterization of depolymerized lignin 3.2.1. Molecular weight analysis The molecular weight distribution curves of EIL and the depolymerized products are shown in Fig. 2. The peaks of the molecular weight distribution curves of the depolymerized products are concentrated in the low-molecular-weight region. Reaction temperatures
Fig. 2. Molecular weight distribution curves of EIL and depolymerized products obtained at reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200).
Fig. 3. FT-IR spectra of EIL and depolymerized products obtained at reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200). 180
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were identified in the spectra of lignin samples, proving that the lignin samples belonged to GHS lignin (Brahim et al., 2017). The intensities of the above-mentioned peaks were similar for different lignin samples, implying the “core” of the lignin structure was not broken during depolymerization. However, there were still several significant differences in the fingerprint region. The spectrum of EIL showed a strong vibration at 1170 cm−1, which is characteristic of conjugated C]O in p-coumaric acid (Jose et al., 2007). However, at the depolymerization temperature of 160 °C, the intensity of the band at 1170 cm−1 became weak, and as reaction temperature increased up to 180 °C and 200 °C, the band eventually disappeared. This indicated that depolymerization damaged the structure of p-coumaric acid, resulting in the absence of the carbonyl group structure. The intensity of the peak around 1326 cm−1 (originating from the CeO stretching of syringyl ring / breathing of syringyl (S) ring plus guaiacyl (G) ring condensed) in the spectrum of D160 was reduced compared with that in the EIL spectrum. This suggested that the ether linkage was broken during depolymerization. Furthermore, this phenomenon was more significant with the increase in S0. The intensity of the signal at 1370 cm-1 stemming from a phenolic OH vibration in the spectrum of the depolymerized product was enhanced, indicating the increase in the phenolic hydroxyl groups after depolymerization. Moreover, the absence of the band at 983 cm-1 (attributed to the vibration of eCH]CH-out-of-plane deformation) and 1630 cm-1 (attributed to ethylene group resonance) in the spectra of D200 (Faix, 1991), which might originate from p-hydroxycinnamyl alcohol units or p-coumaric acid units, suggested that phenolic acid units could be easily removed from lignin macromolecular chains by depolymerization. These observations were further proven by Py-GC/MS and NMR analyses.
dihydrobenzofuran). With the increase in S0, the yield of 2,3-dihydrobenzofuran decreased significantly (from 45.46% in EIL to 13.16% in D-200). Previous work interpreted that 2,3-dihydrobenzofuran was the simplest model compound related to phenylcoumaran linkage (Zakzeski et al., 2010). Meanwhile, as it was difficult to form phenylcoumaran linkage by the syringyl unit without a free Ce5 position in the benzene ring (Toledano et al., 2013), the high proportion of 2,3dihydrobenzofuran represented a high proportion of guaiacyl units and low proportion of syringyl units in the lignin sample. After depolymerization, the phenylcoumaran linkage would be damaged, resulting in the depolymerized lignin possessing a lesser number of heterocyclic rings. Thus, during pyrolysis, the depolymerized lignin would produce less 2,3-dihydrobenzofuran structures but split more phenol and guaiacyl units. This suggests that depolymerization causes the disruption of phenylcoumaran linkages (aryl ether linkages), and the depolymerized products tend to produce phenol units during pyrolysis. It is noteworthy that a large number of vinyl phenol units which originated from p-hydroxycinnamyl alcohol units or p-coumaric acid units were observed in the pyrolyzed products (Fig. 4 and Table S1) (Scholze and Meier, 2001). As confirmed in the FT-IR analysis, the phenolic acid units were easily removed from the lignin chain. Therefore, the number of vinyl phenol units from depolymerized lignin decreased sharply. In addition, the appearance of phenyl and fatty hydrocarbon types indicated the breakage of the side chain of lignin structure units. Furthermore, this phenomenon became more obvious with the increase in S0. This may be because during fast pyrolysis, the low-molecular-weight lignin induces side-chain reactions during pyrolysis, compared to high-molecular-weight lignin. 3.2.4. NMR analysis To interpret the structural features of depolymerized lignin, the D160, D-180, and D-200 samples were investigated by 13C NMR (Fig. S3). Table S2 shows the specific assignments of the correlated lignin signals in the 13C NMR spectra based on a previous literature (Sun et al., 2014). The visible signals in all spectra of depolymerization lignin were due to strong resonances at 56.2 ppm originating from the methoxy group (eOCH3) (Capanema et al., 2004). β-O-4′ linkages without Cα]O (at 60.3 ppm) and with Cα]O (at 63.2 ppm) were observed in the spectra of depolymerized lignin (Li et al., 2012). With the increase in S0, both β-O-4′ linkage types decreased sharply, confirming the cleavage of β-O-4′ linkages via depolymerization. Other typical signals were observed at 166.5, 160.0, 144.7, 130.3, 125.1, 116.0, and 115.0 ppm originating from C9, C4, C7, C2/C6, C1, C3/C5, and C8 in p-coumaric acid, respectively, in the spectrum of D-160 (Ralph et al., 2004). This implied that the composition of lignin used in this experiment contained more p-coumaric acid, different from that in wood species. During the depolymerization reaction, the typical signals from p-coumaric acid apparently weakened in the spectrum of D-180, and even disappeared in the spectrum of D-200. 2D-HSQC NMR provided an essential structural characterization of the lignin, including unit linkages and unit types. The HSQC NMR spectra of the lignin can be divided into two regions-the side-chain regions (δC/δH 50–90/2.5–5.5) the aromatic regions (δC/δH 100–150/ 5.5–8.5), which are displayed in Fig. 5 (Yuan et al., 2011b). The detailed cross signal assignments in the HSQC NMR spectra are shown in Table S3, and the substructures observed in the HSQC NMR spectra are presented in Fig. S4 (Sharazi et al., 2018). In the side-chain regions, signals corresponding to β-O-4′ substructures were observed in the spectrum of D-160, including Cγ−Hγ in β-O-4′ substructures (at 59.2/3.59), Cγ−Hγ in γ-acylated β-O-4′ (at 63.9/4.35), Cα−Hα in β-O-4′ substructures and γ-acylated β-O-4′ substructures (at 71.9/5.10) (Yuan et al., 2011a). However, the signals originating from the β-O-4′ substructures of D-180 and D-200 visibly weakened and even disappeared. This was also confirmed by the results of 13C NMR analysis, and suggested that the β-O-4′ linkages were broken by ethanol/acid catalysis. In addition to β-O-4′ structures,
3.2.3. Py-GC/MS analysis In order to further investigate the main changes in chemical structure of lignin and unit composition, the lignin samples were analyzed by Py-GC/MS. The Py-GC/MS pyrogram of the lignin samples is shown in Fig. S2. The main identified products from the pyrolysis of lignin can be divided into six categories (heterocycle, syringyl types, guaiacyl types, phenol types, phenyl types and fatty hydrocarbon) as shown in Fig. 4 and Table S1 (Lagerquist et al., 2018; Lou et al., 2010a). Trace carbohydrate-derived compounds (2-methylfuran and aliphatic alcohol) are present in the pyrogram of the lignin samples (Camarero et al., 2001), which is in accordance with the composition analysis (Section 3.1). However, there were visible differences in the Py-GC/MS pyrograms among the different lignin samples. The most prominent change was found in the content of the heterocycle (mainly 2,3-
Fig. 4. Distribution of products determined by the Py-GC/MS from EIL and depolymerized products obtained at reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200). 181
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Fig. 5. Side-chain regions (A, C, and E) and aromatic regions (B, D, and F) in the 2D HSQC NMR spectra of depolymerized products: δC/δH 45–75/2.5–6.0 and δC/δH 45–75/2.5–6.0, respectively. D-160 (A, B), D-180 (C, D), D-200 (E, F).
another significant linkage (phenylcoumaran) was also found, including CβeHβ in phenylcoumaran at 53.2/3.60 and CγeHγ in phenylcoumaran at 61.4/3.79 (Wen et al., 2013). With the increase in S0, the phenylcoumaran structures of D-180 and D-200 reduced and disappeared eventually. This supported the Py-GC/MS results that the phenylcoumaran structures were destroyed during the depolymerization reaction, further resulting in a decrease in the amount of 2,3-dihydrobenzofuran. As for the aromatic regions, the main signals in the aromatic region of the lignin samples originated from p-hydroxyphenyl, syringyl, and guaiacyl units. The prominent signals from guaiacyl units were 111.2/ 7.34 (C2eH2 in oxidized (Cα]O) guaiacyl units), 115.6/6.82 (C2eH2 in guaiacyl units), and 118.8/6.85 (C6eH6 in guaiacyl units) (Yuan et al., 2011b). During depolymerization, the structure of the guaiacyl units showed no visible changes. The signals originating from C2,6eH2,6 in etherified syringyl (S) units (at 103.8/6.71) were weak in the spectra of all lignin samples, which suggested a small number of S units. This
agreed with the conclusion of the Py-GC/MS analysis. The signal of the p-hydroxyphenyl (H) unit was also observed at 127.9/7.19 (Yin et al., 2017). In addition, p-coumaric acid showed a series of considerable signals for C3,5-H3,5 at 122.8/7.14, C2,6-H2,6 at 130.2/7.55, and Cα-Hα at 144.8/7.57 in the spectra of D-160 and D-180 (Rencoret et al., 2009). With the increase in the reaction temperature up to 200 °C, these signals of p-coumaric acid were weakened and eventually disappeared, which agreed with the results of FT-IR, Py-GC/MS, and 13C NMR analyses. The acid-catalyzed depolymerization of lignin in ethanol solvent can be interpreted based on previous literature (Li et al., 2015; Lou et al., 2010b; Voitl and Rudolf von Rohr, 2008; Zakzeski et al., 2010). The model structure of lignin in this work is given in Fig. 6. The dominant change during depolymerization is the breakage of the β-O-4′ linkages. Other visible changes occur in the phenylcoumaran structure. Phenylcoumaran is the linkage of heterocycles, and is formed by β-5′ and αO-4′. During depolymerization, the α-O-4′ of the phenylcoumaran is readily hydrolyzed, resulting in the disruption of phenylcoumaran 182
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Fig. 6. Structural transformation of lignin during moderate ethanol/acid catalysis depolymerization.
structure. In addition, p-coumaric acid, which is typically connected to lignin by ester linkages, is dissolved owing to the breaking of ester linkages in the acid system. These hypotheses also additionally supply reasonable interpretations for the results of FT-IR, Py-GC/MS, 13C NMR, and HSQC NMR analyses. 3.3. Antioxidant activity of depolymerized lignin In order to explore the antioxidant activity of the depolymerized lignin, the total phenolics content, which is positively correlated to the antioxidant activity of lignin, was determined (An et al., 2017). As shown in Table 3, the depolymerized products at different reaction temperatures exhibited significant changes in the total phenolics content. The total phenolics contents of D-160, D-180 and D-200 were 145.9 ± 4.5, 149.9 ± 4.8 and 167.0 ± 3.6 mg GAE/g, respectively, and were visibly elevated compared to that of EIL (105.7 ± 4.6 mg GAE/g). Moreover, with the increase in S0, the total phenolics content further increased, which agreed with the decrease in molecular weight (Barapatre et al., 2015). Based on the above structural characterizations, the antioxidant
Fig. 7. DPPH free radical scavenging capacity of EIL and depolymerized products obtained at reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200). BHT was used as a positive control.
activities of the depolymerized products were further evaluated by a DPPH free radical scavenging assay. DPPH free radical scavenging capacities and IC50 values of all lignin samples are listed in Fig. 7 and Table 3, respectively. As shown in Table 3, the IC50 of D-160 (92.8 ± 3.6 μg/mL) was obviously reduced compared with the IC50 of EIL (168.1 ± 2.2 μg/mL), which implied that ethanol/acid catalysis resulted in the improvement of antioxidant activity. In addition, with the reaction temperature increasing up to 180 °C and 200 °C, the depolymerized products (D-180 and D-200) with lower molecular weight and higher total phenolics content exhibited lower IC50 values (83.9 ± 4.1 μg/mL and 66.0 ± 2.6 μg/mL, respectively). Although the depolymerized products (D-180 and D-200) possessed similar molecular weight, the DPPH free radical scavenging capacities of D-180 and D-200 were significantly different, possibly because D-200 has a lower polydispersity index and higher solubility (Dizhbite et al., 2004; Pan et al., 2006). Notably, the DPPH radical scavenging capacity of D-200 (IC50 = 66.0 ± 2.6 μg/mL) was close to that of the positive control
Table 3 Total phenolics content and DPPH free radical scavenging capacities (IC50 value) of EIL and depolymerized products obtained with reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200). Sample/condition
Total phenolics content/ (mg GAE/g)a*
IC50 (μg/mL)b*
EIL / 0 °C D-160/160 °C D-180/180 °C D-200/200 °C BHT
105.7 145.9 149.9 167.0 –
168.1 ± 2.2 92.8 ± 3.6 83.9 ± 4.1 66.0 ± 2.6 53.3 ± 4.5
± ± ± ±
4.6 4.5 4.8 3.6
a*: The total phenolics content of the sample was expressed as gallic acid equivalent (GAE). The calibration curve of gallic acid in DMSO (with six different concentrations from 4 μg/mL-20 μg/mL) was drawn in advance. b*: For a more intuitive display of the results, the IC50 values (the concentration required with 50% inhibition of radical formation) were further calculated based on the RSA with different sample concentrations. 183
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BHT (IC50 = 53.3 ± 4.5 μg/mL), indicating that the lignin sample after depolymerization has promising potential for the application as natural antioxidants. These results suggest that the cleavage of lignin ether linkages by ethanol/acid catalysis shortens the polymer chain and enhances the total phenolics content and antioxidant activity.
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4. Conclusion The moderate depolymerization of high-molecular-weight lignin was conducted in ethanol/acid catalysis system. Depolymerization was caused by the cleavage of the ether linkages, which was confirmed by the results of GPC, FT-IR, Py-GC/MS, 13C NMR, and HSQC NMR analyses, and resulting in a decrease in the molecular weight and enhancement of lignin solubility. The existence of ethanol might play an important role in suppressing lignin condensation. Simultaneously, the total phenolics contents of the depolymerized products (D-160, D-180, and D-200) were enhanced sharply. The antioxidant activities of the depolymerized products were improved significantly. Particularly, the DPPH free radical scavenging capacity of D-200 was close to that of BHT, which indicated that lignin is a promising candidate as a commercial antioxidant. Consequently, our work confirmed that moderate depolymerization by in situ ethanol/acid catalysis is an effective way to improve the solubility and antioxidant activity of lignin. Acknowledgments This work was kindly supported by the National Key Research and Development Program of China (Grant No. 2017YFB0307903), National Natural Science Foundation of China (31700515), Natural Science Foundation of Tianjin City (16JCQNJC05900) and Research Plan Project of Tianjin Municipal Education Commission (2017KJ023), State Key Laboratory of Pulp and Paper Engineering (201822, 201503, 201451), Tianjin Key Laboratory of Pulp & Paper (201607) and Key Laboratory of Pulp and Paper Science & Technology of the Ministry of Education of China (KF2015014). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2018.11.009. References Aminzadeh, S., Lauberts, M., Dobele, G., Ponomarenko, J., Mattsson, T., Lindström, M.E., Sevastyanova, O., 2018. Membrane filtration of kraft lignin: Structural charactristics and antioxidant activity of the low-molecular-weight fraction. Ind. Crop. Prod. 112, 200–209. An, L., Wang, G., Jia, H., Liu, C., Sui, W., Si, C., 2017. Fractionation of enzymatic hydrolysis lignin by sequential extraction for enhancing antioxidant performance. Int. J. Biol. Macromol. 99, 674–681. Arni, S.A., 2018. Extraction and isolation methods for lignin separation from sugarcane bagasse: a review. Ind. Crop. Prod. 115, 330–339. Barapatre, A., Aadil, K.R., Tiwary, B.N., Jha, H., 2015. In vitro antioxidant and antidiabetic activities of biomodified lignin from Acacia nilotica wood. Int. J. Biol. Macromol. 75, 81–89. Brahim, M., Boussetta, N., Grimi, N., Vorobiev, E., Zieger-Devin, I., Brosse, N., 2017. Pretreatment optimization from rapeseed straw and lignin characterization. Ind. Crop. Prod. 95, 643–650. Camarero, S., Bocchini, P., Galletti, G.C., Martı́nez, M.J., Martı́nez, A.T., 2001. Compositional changes of wheat lignin by a fungal peroxidase analyzed by pyrolysisGC-MS. J. Anal. Appl. Pyrol. 58–59, 413–423. Capanema, E.A., Balakshin, M.Y., Kadla, J.F., 2004. A comprehensive approach for quantitative lignin characterization by NMR spectroscopy. J. Agric. Food Chem. 52, 1850–1860. Dai, L., Liu, R., Hu, L.Q., Zou, Z.F., Si, C.L., 2017. Lignin nanoparticle as a novel green carrier for the efficient delivery of resveratrol. ACS Sustain. Chem. Eng. 5, 8241–8249. Dai, L., Liu, R., Si, C., 2018. A novel functional lignin-based filler for pyrolysis and feedstock recycling of poly(l-lactide). Green Chem. 20, 1777–1783. Deuss, P.J., Scott, M., Tran, F., Westwood, N.J., de Vries, J.G., Barta, K., 2015. Aromatic monomers by in situ conversion of reactive intermediates in the acid-catalyzed depolymerization of lignin. J. Am. Chem. Soc. 137, 7456–7467.
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Wang, G., Xia, Y., Sui, W., Si, C., 2018c. Lignin as a novel tyrosinase Inhibitor: effects of sources and isolation processes. ACS Sustain. Chem. Eng. 6, 9510–9518. Wen, J.L., Xue, B.L., Xu, F., Sun, R.C., Pinkert, A., 2013. Unmasking the structural features and property of lignin from bamboo. Ind. Crop. Prod. 42, 332–343. Yin, H.S., Liu, H.M., Liu, Y.L., 2017. Structural characterization of lignin in fruits and stalks of Chinese quince. Molecules 22. Yuan, T.Q., Sun, S.N., Xu, F., Sun, R.C., 2011a. Characterization of lignin structures and lignin–carbohydrate complex (LCC) linkages by quantitative 13C and 2D HSQC NMR spectroscopy. J. Agric. Food Chem. 59, 10604–10614. Yuan, T.Q., Sun, S.N., Xu, F., Sun, R.C., 2011b. Structural characterization of lignin from triploid of populus tomentosa carr. J. Agric. Food Chem. 59, 6605–6615. Zakzeski, J., Jongerius, A.L., Weckhuysen, B.M., 2010. Transition metal catalyzed oxidation of Alcell lignin, soda lignin, and lignin model compounds in ionic liquids. Green Chem. 12, 1225–1236. Zikeli, F., Ters, T., Fackler, K., Srebotnik, E., Li, J., 2016. Fractionation of wheat straw Dioxane lignin reveals molar mass dependent structural differences. Ind. Crop. Prod. 91, 186–193.
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Enhancing the solubility and antioxidant activity of high-molecular-weight lignin by moderate depolymerization via in situ ethanol/acid catalysis
T
Liangliang Ana, Chuanling Sia,b, , Guanhua Wanga,b,c, , Wenjie Suid, Zhengyi Taoa,b ⁎
⁎
a
Tianjin Key Laboratory of Pulp & Paper, College of Papermaking Science and Technology, Tianjin University of Science & Technology, Tianjin 300457, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China c Key Laboratory of Pulp and Paper Science & Technology of Ministry of Education of China, Qilu University of Technology, Jinan 250353, China d Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, China b
ARTICLE INFO
ABSTRACT
Keywords: Enzymatic hydrolysis lignin Mild depolymerization Antioxidant activity Solubility
Lignin, a by-product obtained from the pulping industry and cellulosic ethanol production, has received considerable attention for its antioxidant activity owing to its polyphenol structure. In this work, high-molecularweight lignin obtained by ethanol-water fractionation of enzymatic hydrolysis lignin, was moderately depolymerized via in situ ethanol/acid catalysis to improve its solubility and antioxidant activity. The depolymerized products showed significant reduction in molecular weight and improvement in solubility in an 80% ethanolwater (v/v) solution owing to the cleavage of ether linkages; this was confirmed by gel permeation chromatography (GPC), fourier transform-infrared spectroscopy (FT-IR), pyrolysis-gas chromatography/ mass spectrometer (Py-GC/MS), carbon-13 nuclear magnetic resonance spectrometry (13C NMR), and heteronculear single quantum coherence nuclear magnetic resonance spectrometry (HSQC NMR) analyses. In addition, the total phenolics content of the depolymerized products increased sharply, leading to a significant enhancement in the free radical scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl (DPPH). In particular, D-200 (depolymerized product at 200 °C) with IC50 = 66.0 ± 2.6 μg/mL was comparable to the positive control butylated hydroxytoluene (BHT) with IC50 = 53.3 ± 4.5 μg/mL, indicating the potential application of lignin as a high-valueadded antioxidant. In conclusion, the depolymerization of high-molecular-weight lignin via moderate ethanol/ acid catalysis may effectively improve its solubility and antioxidant activity.
1. Introduction Lignin, a heterogeneous natural polymer, consists of C6-C3 phenyl propane units including coniferyl alcohol (G), sinapyl alcohol (S), and p-coumaryl alcohol (H), and typically holds about 15–30% by weight and about 40% by energy in biomass (Doherty et al., 2011). Despite its enormous reserves and energy storage, lignin is underutilized in the current cellulose-dominant processing industries and is typically burned to generate power directly. Particularly with cellulosic ethanol production approaching commercial practices, immense quantities of enzymatic hydrolysis lignin are produced as a by-product. In addition, owing to the little use of other biomass components except for cellulose, the path of cellulosic ethanol is less economically competitive. Therefore, the high-value-added utilization of lignin plays a significant role in the commercial operation of lignocellulosic bio-refinery (Dai et al., 2017; Li et al., 2015).
As a natural and renewable compound with a polyphenol structure, lignin shows promising antioxidant activity, and several studies have focused on the exploration of its antioxidant properties (Tang et al., 2017; Wang et al., 2018c). García et al. evaluated the antioxidant activities of 14 lignin samples and discovered that autohydrolysis, organosolv, and alkali lignin exhibited similar antioxidant activities with natural antioxidant (García et al., 2012). With the advancement in research, the relationship between the structure and antioxidant capacity of lignin was further studied (Pan et al., 2006; Sun et al., 2014). It has been established that high molecular weight is a crucial factor that decreases the radical scavenging capacity of lignin (Aminzadeh et al., 2018). The incompatibility caused by poor solubility is also a vital issue that needs to be addressed (Laurichesse and Avérous, 2014). In our previous work, we demonstrated that lignin with low molecular weight and high total phenolics content exhibited preferable antioxidant activity (An et al., 2017). Therefore, obtaining low-molecular-weight
⁎ Corresponding authors at: Tianjin Key Laboratory of Pulp & Paper, College of Papermaking Science and Technology, Tianjin University of Science & Technology, Tianjin 300457, China. E-mail addresses: [email protected] (C. Si), [email protected] (G. Wang).
https://doi.org/10.1016/j.indcrop.2018.11.009 Received 10 April 2018; Received in revised form 13 September 2018; Accepted 3 November 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Extraction, fractionation, and depolymerization of enzymatic hydrolysis lignin (EHL).
lignin, increasing the total phenolics content, and improving the lignin solubility are considered feasible ways to improve its antioxidant activity. The selective acid-catalyzed depolymerization of lignin to obtain low-molecular-weight products has attracted immense interest in recent years. Lignin depolymerization by acid catalysis is a widely used method that breaks ether linkages to offer small segments including oligomeric and monomeric aromatic products (Li et al., 2015; Wang et al., 2018a). Although acid-catalyzed depolymerization of lignin in an aqueous system is a feasible technique to generate low-molecularweight products, the yield of the target products is often low owing to lignin condensation. Therefore, there have been attempts to inhibit condensation (Deuss et al., 2015; Güvenatam et al., 2014; Huang et al., 2015). For example, Ma et al. investigated the depolymerization of kraft lignin with multiple catalytic agents and noted that ethanol was an effective solvent in suppressing condensation and char-forming reactions (Ma et al., 2014). However, these studies mainly focused on identifying the promising substitutes of phenyl chemicals and liquid fuels owing to the growing shortage of fossil energy (Li et al., 2015), and less research has been performed on the relationship between lignin depolymerization and antioxidant activity. Thus, the hypothesis of this work is that the lignin antioxidant activity, which has a negative correlation with the molecular weight, may be enhanced by depolymerization owing to the decrease of molecular weight. In order to improve antioxidant activity, the high-molecularweight lignin part (insoluble in 80% ethanol) from the fractionation of enzymatic hydrolysis lignin was depolymerized in situ in 80% ethanol solvent via acid catalysis. Here, ethanol as a relatively cheap solvent from biomass bio-conversion, was used as the solvent for fractionation and depolymerization. The depolymerized products were characterized by gel permeation chromatography (GPC), fourier transform-infrared
spectroscopy (FT-IR), pyrolysis-gas chromatography/ mass spectrometer (Py-GC/MS), carbon-13 nuclear magnetic resonance spectrometry (13C NMR), and heteronculear single quantum coherence nuclear magnetic resonance spectrometry (HSQC NMR). The total phenolics content of the lignin samples was determined, and the free radical scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) was further measured to confirm the effect of the depolymerization on the antioxidant activity of lignin. 2. Material and methods 2.1. Materials Enzymatic hydrolysis residues (EHRs) of corn straw produced from steam explosion pretreatment and cellulosic enzymatic hydrolysis were kindly provided by Songyuan Laihe Chemical Co. Ltd, Jilin Province, China. All commercial chemicals were analytical reagents. 2.2. Extraction, fractionation, and depolymerization of lignin Enzymatic hydrolysis lignin (EHL) was isolated from EHRs by leaching in an aqueous solution of 1,4-dioxane (9:1, v/v), with a solid to liquid ratio of 1:10 (g/mL), assisted with a magnetic stirrer at room temperature for 12 h. After extraction, the mixture was centrifuged and the supernatant was concentrated by rotary evaporation to remove the solvent. After the addition of distilled water, the precipitate (EHL) was collected via centrifugation (Zikeli et al., 2016). Subsequently, 10 g EHL was added to 500 mL 80% ethanol-water solution, and the mixture was stirred with a magnetic stirrer at room temperature for 1 h followed by paper filtration (Wang and Chen, 2013a). The ethanol-insoluble lignin (EIL) was collected and then 178
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Table 1 Chemical composition of lignin samples: EHRs: enzymatic hydrolysis residues; EHL: enzymatic hydrolysis lignin. Lignin samples
Lignin/%
EHRs EHL
Carbohydrate/%
Acid-insoluble lignin
Acid-soluble lignin
Glucose
Xylose
Arabinose
52.20 ± 1.69 82.62 ± 1.05
2.38 ± 0.06 3.72 ± 0.12
20.55 ± 0.26 2.75 ± 0.69
6.88 ± 0.27 3.76 ± 0.19
0.73 ± 0.00 0.70 ± 0.06
depolymerized in situ in 80% ethanol. The depolymerization was conducted in a 150 mL stainless steel batch reactor. For this, 1.5 g EIL was suspended in 75 mL 80% ethanolwater solution (v/v, 4:1) that contained 0.10 mL 98% H2SO4, and the reaction mixture was heated to the desired reaction temperature under continuous stirring at 225 rpm for 1 h. After the reaction, the reactor was rapidly quenched with flowing water. The reaction mixture was filtered, and the filter cake (reaction residues) was dried to a constant weight. The filtrate was concentrated to a volume of 10 mL, followed by the addition of distilled water to precipitate product. The precipitant was washed to neutral pH with distilled water and collected as the depolymerized product. The flow-process diagram of extraction, fractionation, and depolymerization of lignin is shown in Fig. 1. The conversion rate of depolymerization was calculated by formula (1), and the yield of depolymerized products was calculated by formula (2):
conversion (%) =
Yield (%) =
mp mn
mn mr mn
× 100%
× 100%
100)/14.75*t )
11.25 ± 1.62 1.36 ± 0.00
dissolved in 0.5 mL dimethylsulfoxide-d6 (99.9%, Cambridge Isotopes Laboratories) to record the 13C NMR and HSQC NMR spectra (Sun et al., 2012). A single-shot pyrolyzer (Py-2020iS, Japan) coupled with a GC/MS (Agilent Technologies 5975C, America) i.e., a Py-GC/MS was employed to analyze the lignin structure according to literature (Dai et al., 2018; Liu et al., 2018). 2.4. Determination of the total phenolics content and antioxidant activity The total phenolics content of lignin was determined by the FolinCiocalteu (F-C) method (An et al., 2017). The DPPH free radical scavenging capacities for the lignin samples were evaluated in accordance with the protocol described in a previous literature (Lauberts et al., 2017).
(1)
3. Results and discussion 3.1. Lignin extraction, fractionation, and depolymerization
(2)
In order to minimize the destruction of the EHL structure during the extraction process, a mild method (using neutral 1,4-dioxane solvent at room temperature) was applied. After a quantitative comparison of lignin and polysaccharide content changes in the samples (Table 1), it was concluded that the mild extraction process was an effective way to provide a good substrate for the further depolymerization process. The total lignin content increased from 54.58% (EHRs) to 86.33% (EHL) and the increase in acid-insoluble lignin content was the dominant factor that elevated the purity of lignin. The content of acid-insoluble lignin in EHL increased to 82.62%, compared with 52.20% in EHRs. However, the content of acid-soluble lignin in EHL and EHRs did not show visible changes, which may be due to the assumption that the mild neutral solvent extraction process could effectively dissolve lignin and protect the chemical structure of lignin from destruction (Arni, 2018); thus, the acid-soluble lignin still occupied a small proportion of the whole lignin. In addition, the glucose content (2.75%) of EHL decreased sharply, and xylose and arabinose contents of EHL also decreased to 3.76% and 0.70%, respectively. Nevertheless, it was observed that the xylose content was higher than the content of glucose in EHL, which suggested that hemicellulose bonded covalently to lignin to form a lignin carbohydrate complex (LCC) was more difficult to remove during purification. The result agrees well with earlier studies (Lauberts et al., 2017; Wang and Chen, 2014). Next, the purified EHL was fractionated into two fractions by dissolution in 80% ethanol (shown in Fig. 1). The molecular weight distribution curves of EHL and EIL are presented in Fig. S1. From the chromatograms, the molecular weight curve of EIL apparently migrated to the high-molecular-weight section, while the low-molecular-weight section was distinctly weakened. The results indicated that the molecular weight of EIL was markedly higher than that of EHL. This corresponded well with the average molecular weight of EHL (M n = 5265 g/mol, M w = 11,595 g/mol, M w /M n = 2.20) and EIL (M n = 7731 g/mol, M w = 14,218 g/mol, M w /M n = 1.84). With a similar procedure, in our previous investigation, we successfully obtained alkaline-extracted lignin with high molecular weight from steam-exploded corn stalk through ethanol-water fractionation (Wang
where mn is the mass of ethanol-insoluble lignin for the depolymerization reaction in grams; mr is the mass of the reaction residues in grams; mp is the mass of depolymerized products in grams. The depolymerization effect was presented in relation to the “severity factor”, which combines the effects of treatment processing parameters (reaction temperature and reaction time) into a single equation. The severity factor, S0, for each treatment was calculated according to formula (3) (Martin-Sampedro et al., 2011).
S0 = log(e (T
Ash/%
(3)
where T is the reaction temperature in °C; t is the duration of the treatment in minutes. 2.3. Lignin characterization The lignin and carbohydrate contents of EHRs and EHL were determined using the two-step acid hydrolysis method (Sluiter et al., 2008; Wang and Chen, 2016). A modified GPC method using hydrophilic gel column (TSK G3000PWxl column) was employed to analyze the average molecular weight of the lignin samples according to literature (Wang and Chen, 2013b; Wang et al., 2018b). Lignin samples were dissolved in 1% NaOH and diluted with tris-acetate buffer (20 mmol/L, pH 7.4). The column was operated at 25 °C and eluted with tris-acetate buffer at a flow rate of 0.5 mL/min. subsequently, 20 μL samples were injected each time, and a UV-detector (280 nm) was used for detection. The molecular weight was calculated using the Agilent GPC data analysis system. FT-IR analysis was conducted by KBr pressing disc technique in the range of 4000-400 cm−1 using an FT-IR spectrophotometer (FT-IR-650, Gangdong Sci. & Tech. Co., Ltd, China) (Liu et al., 2017). 13 C NMR and HSQC NMR spectra were obtained using a Bruker AVANCE III 400 MHz spectrometer with a 5 mm high-resolution liquid probe. In this study, the lignin obtained via 1,4-dioxane-water solution extraction was readily dissolved in the DMSO-d6 solvent. According to the procedure, approximately 100 mg of the lignin sample was directly 179
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Table 2 Yield of depolymerized products under various temperatures *, and molecular weights of lignin.
* *
Run
Reaction temperature (°C)
S0*
Conversion rate of Depolymerization (%)
Yield of depolymerized products (%)
M n (g/mol)
M w (g/mol)
M w /M n (polydispersity index)
0 0 1 2 3
EHL* EIL* 160 180 200
– – 3.54 4.13 4.72
– – 94.29 94.89 96.69
– – 71.19 67.62 63.31
5265 7731 4742 3071 2856
11,595 14,218 8483 4906 4773
2.20 1.84 1.79 1.60 1.57
Reaction conditions: 80% ethanol-insoluble lignin 1.5 g, 80% ethanol solution 75 mL, 98% H2SO4 solution 0.1 mL, time = 1 h. EHL: enzymatic hydrolysis lignin. * EIL: ethanol-insoluble lignin. * S0: severity factor.
and Chen, 2013a). In order to improve the antioxidant activity of EIL, depolymerization by acid catalysis was performed in 80% ethanol solution (Fig. 1). Table 2 summarizes the yields of the depolymerized products (based on the average results of three parallel experiments). Here, the conversion rate of depolymerization represented the changes of lignin solubility in 80% ethanol solution. The severity factor (S0) increased from 3.54 (reaction temperature at 160 °C) to 4.13 (reaction temperature at 180 °C) and further to 4.72 (reaction temperature at 200 °C). When the reaction temperature was 160 °C, the conversion rate of depolymerization was 94.29%, which indicated that 94.29% of EIL was converted into a dissolvable form in 80% ethanol solution. Meanwhile, with the increase in S0, the conversion was also slightly enhanced, suggesting that the higher reaction temperature tended to promote the solubilization of lignin. However, with the increase in S0, the yield of the depolymerized products decreased. This phenomenon was possibly because the depolymerized lignin was further cracked down to lowermolecular-weight products that were hard to recover (Huang et al., 2015; Wang and Chen, 2013a). In summary, EIL may be depolymerized at a moderate temperature, and the depolymerized products were readily dissolved in 80% ethanol.
had a substantial effect on the molecular weight distribution of lignin. With the increase in S0, the molecular weight distribution curve further shifted towards the low-molecular-weight regions. At 200 °C, 96.69% of EIL was dissolvable in 80% ethanol-water solution, and the depolymerized product had the lowest molecular weight (M n = 2856 g/mol, M w = 4773 g/mol) and narrow polydispersity (M w /M n = 1.57) (Table 2). Compared to lignin depolymerization at higher temperatures (250–350 °C) in previous studies (Güvenatam et al., 2014; Ma et al., 2014), the reaction conditions in this work were comparatively moderate. In addition, as shown in the molecular weight distribution curves of the depolymerized products at different temperatures, lignin can be depolymerized effectively, and the depolymerized products were readily dissolvable in ethanol/water mixtures by moderate catalysis without the formation of agglomerates. This may be because ethanol could generate radicals as capping agents to eliminate the intermediate carbonium ions that are critical for lignin condensation (Huang et al., 2015; Voitl and Rudolf von Rohr, 2008). Hence, the GPC results of this work indicated that the moderate ethanol/acid catalysis can depolymerize high-molecular-weight lignin effectively and decrease the molecular weight significantly. 3.2.2. FT-IR analysis Nondestructive structural characterization by FT-IR was performed to interpret the chemical structure changes of lignin after depolymerization. The hydroxyl groups from phenolic and aliphatic groups at 3410 cm−1 appeared in all spectra (shown in Fig. 3) and a band centered at 2930 cm−1originating from the CeH-stretching in aromatic methoxyl groups, methyl, and the methylene group of side chains was also present (Rashid et al., 2018). In addition, aromatic skeleton vibrations were observed at 1600 cm−1, 1515 cm−1 and 1426 cm−1 in all lignin spectra. Peaks at 1260 cm−1 (guaiacyl unit, G), 1125 cm−1 (syringyl unit, S) and 834 cm−1 (CeH out-of-plane in S and H units)
3.2. Molecular weight and structural characterization of depolymerized lignin 3.2.1. Molecular weight analysis The molecular weight distribution curves of EIL and the depolymerized products are shown in Fig. 2. The peaks of the molecular weight distribution curves of the depolymerized products are concentrated in the low-molecular-weight region. Reaction temperatures
Fig. 2. Molecular weight distribution curves of EIL and depolymerized products obtained at reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200).
Fig. 3. FT-IR spectra of EIL and depolymerized products obtained at reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200). 180
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were identified in the spectra of lignin samples, proving that the lignin samples belonged to GHS lignin (Brahim et al., 2017). The intensities of the above-mentioned peaks were similar for different lignin samples, implying the “core” of the lignin structure was not broken during depolymerization. However, there were still several significant differences in the fingerprint region. The spectrum of EIL showed a strong vibration at 1170 cm−1, which is characteristic of conjugated C]O in p-coumaric acid (Jose et al., 2007). However, at the depolymerization temperature of 160 °C, the intensity of the band at 1170 cm−1 became weak, and as reaction temperature increased up to 180 °C and 200 °C, the band eventually disappeared. This indicated that depolymerization damaged the structure of p-coumaric acid, resulting in the absence of the carbonyl group structure. The intensity of the peak around 1326 cm−1 (originating from the CeO stretching of syringyl ring / breathing of syringyl (S) ring plus guaiacyl (G) ring condensed) in the spectrum of D160 was reduced compared with that in the EIL spectrum. This suggested that the ether linkage was broken during depolymerization. Furthermore, this phenomenon was more significant with the increase in S0. The intensity of the signal at 1370 cm-1 stemming from a phenolic OH vibration in the spectrum of the depolymerized product was enhanced, indicating the increase in the phenolic hydroxyl groups after depolymerization. Moreover, the absence of the band at 983 cm-1 (attributed to the vibration of eCH]CH-out-of-plane deformation) and 1630 cm-1 (attributed to ethylene group resonance) in the spectra of D200 (Faix, 1991), which might originate from p-hydroxycinnamyl alcohol units or p-coumaric acid units, suggested that phenolic acid units could be easily removed from lignin macromolecular chains by depolymerization. These observations were further proven by Py-GC/MS and NMR analyses.
dihydrobenzofuran). With the increase in S0, the yield of 2,3-dihydrobenzofuran decreased significantly (from 45.46% in EIL to 13.16% in D-200). Previous work interpreted that 2,3-dihydrobenzofuran was the simplest model compound related to phenylcoumaran linkage (Zakzeski et al., 2010). Meanwhile, as it was difficult to form phenylcoumaran linkage by the syringyl unit without a free Ce5 position in the benzene ring (Toledano et al., 2013), the high proportion of 2,3dihydrobenzofuran represented a high proportion of guaiacyl units and low proportion of syringyl units in the lignin sample. After depolymerization, the phenylcoumaran linkage would be damaged, resulting in the depolymerized lignin possessing a lesser number of heterocyclic rings. Thus, during pyrolysis, the depolymerized lignin would produce less 2,3-dihydrobenzofuran structures but split more phenol and guaiacyl units. This suggests that depolymerization causes the disruption of phenylcoumaran linkages (aryl ether linkages), and the depolymerized products tend to produce phenol units during pyrolysis. It is noteworthy that a large number of vinyl phenol units which originated from p-hydroxycinnamyl alcohol units or p-coumaric acid units were observed in the pyrolyzed products (Fig. 4 and Table S1) (Scholze and Meier, 2001). As confirmed in the FT-IR analysis, the phenolic acid units were easily removed from the lignin chain. Therefore, the number of vinyl phenol units from depolymerized lignin decreased sharply. In addition, the appearance of phenyl and fatty hydrocarbon types indicated the breakage of the side chain of lignin structure units. Furthermore, this phenomenon became more obvious with the increase in S0. This may be because during fast pyrolysis, the low-molecular-weight lignin induces side-chain reactions during pyrolysis, compared to high-molecular-weight lignin. 3.2.4. NMR analysis To interpret the structural features of depolymerized lignin, the D160, D-180, and D-200 samples were investigated by 13C NMR (Fig. S3). Table S2 shows the specific assignments of the correlated lignin signals in the 13C NMR spectra based on a previous literature (Sun et al., 2014). The visible signals in all spectra of depolymerization lignin were due to strong resonances at 56.2 ppm originating from the methoxy group (eOCH3) (Capanema et al., 2004). β-O-4′ linkages without Cα]O (at 60.3 ppm) and with Cα]O (at 63.2 ppm) were observed in the spectra of depolymerized lignin (Li et al., 2012). With the increase in S0, both β-O-4′ linkage types decreased sharply, confirming the cleavage of β-O-4′ linkages via depolymerization. Other typical signals were observed at 166.5, 160.0, 144.7, 130.3, 125.1, 116.0, and 115.0 ppm originating from C9, C4, C7, C2/C6, C1, C3/C5, and C8 in p-coumaric acid, respectively, in the spectrum of D-160 (Ralph et al., 2004). This implied that the composition of lignin used in this experiment contained more p-coumaric acid, different from that in wood species. During the depolymerization reaction, the typical signals from p-coumaric acid apparently weakened in the spectrum of D-180, and even disappeared in the spectrum of D-200. 2D-HSQC NMR provided an essential structural characterization of the lignin, including unit linkages and unit types. The HSQC NMR spectra of the lignin can be divided into two regions-the side-chain regions (δC/δH 50–90/2.5–5.5) the aromatic regions (δC/δH 100–150/ 5.5–8.5), which are displayed in Fig. 5 (Yuan et al., 2011b). The detailed cross signal assignments in the HSQC NMR spectra are shown in Table S3, and the substructures observed in the HSQC NMR spectra are presented in Fig. S4 (Sharazi et al., 2018). In the side-chain regions, signals corresponding to β-O-4′ substructures were observed in the spectrum of D-160, including Cγ−Hγ in β-O-4′ substructures (at 59.2/3.59), Cγ−Hγ in γ-acylated β-O-4′ (at 63.9/4.35), Cα−Hα in β-O-4′ substructures and γ-acylated β-O-4′ substructures (at 71.9/5.10) (Yuan et al., 2011a). However, the signals originating from the β-O-4′ substructures of D-180 and D-200 visibly weakened and even disappeared. This was also confirmed by the results of 13C NMR analysis, and suggested that the β-O-4′ linkages were broken by ethanol/acid catalysis. In addition to β-O-4′ structures,
3.2.3. Py-GC/MS analysis In order to further investigate the main changes in chemical structure of lignin and unit composition, the lignin samples were analyzed by Py-GC/MS. The Py-GC/MS pyrogram of the lignin samples is shown in Fig. S2. The main identified products from the pyrolysis of lignin can be divided into six categories (heterocycle, syringyl types, guaiacyl types, phenol types, phenyl types and fatty hydrocarbon) as shown in Fig. 4 and Table S1 (Lagerquist et al., 2018; Lou et al., 2010a). Trace carbohydrate-derived compounds (2-methylfuran and aliphatic alcohol) are present in the pyrogram of the lignin samples (Camarero et al., 2001), which is in accordance with the composition analysis (Section 3.1). However, there were visible differences in the Py-GC/MS pyrograms among the different lignin samples. The most prominent change was found in the content of the heterocycle (mainly 2,3-
Fig. 4. Distribution of products determined by the Py-GC/MS from EIL and depolymerized products obtained at reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200). 181
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Fig. 5. Side-chain regions (A, C, and E) and aromatic regions (B, D, and F) in the 2D HSQC NMR spectra of depolymerized products: δC/δH 45–75/2.5–6.0 and δC/δH 45–75/2.5–6.0, respectively. D-160 (A, B), D-180 (C, D), D-200 (E, F).
another significant linkage (phenylcoumaran) was also found, including CβeHβ in phenylcoumaran at 53.2/3.60 and CγeHγ in phenylcoumaran at 61.4/3.79 (Wen et al., 2013). With the increase in S0, the phenylcoumaran structures of D-180 and D-200 reduced and disappeared eventually. This supported the Py-GC/MS results that the phenylcoumaran structures were destroyed during the depolymerization reaction, further resulting in a decrease in the amount of 2,3-dihydrobenzofuran. As for the aromatic regions, the main signals in the aromatic region of the lignin samples originated from p-hydroxyphenyl, syringyl, and guaiacyl units. The prominent signals from guaiacyl units were 111.2/ 7.34 (C2eH2 in oxidized (Cα]O) guaiacyl units), 115.6/6.82 (C2eH2 in guaiacyl units), and 118.8/6.85 (C6eH6 in guaiacyl units) (Yuan et al., 2011b). During depolymerization, the structure of the guaiacyl units showed no visible changes. The signals originating from C2,6eH2,6 in etherified syringyl (S) units (at 103.8/6.71) were weak in the spectra of all lignin samples, which suggested a small number of S units. This
agreed with the conclusion of the Py-GC/MS analysis. The signal of the p-hydroxyphenyl (H) unit was also observed at 127.9/7.19 (Yin et al., 2017). In addition, p-coumaric acid showed a series of considerable signals for C3,5-H3,5 at 122.8/7.14, C2,6-H2,6 at 130.2/7.55, and Cα-Hα at 144.8/7.57 in the spectra of D-160 and D-180 (Rencoret et al., 2009). With the increase in the reaction temperature up to 200 °C, these signals of p-coumaric acid were weakened and eventually disappeared, which agreed with the results of FT-IR, Py-GC/MS, and 13C NMR analyses. The acid-catalyzed depolymerization of lignin in ethanol solvent can be interpreted based on previous literature (Li et al., 2015; Lou et al., 2010b; Voitl and Rudolf von Rohr, 2008; Zakzeski et al., 2010). The model structure of lignin in this work is given in Fig. 6. The dominant change during depolymerization is the breakage of the β-O-4′ linkages. Other visible changes occur in the phenylcoumaran structure. Phenylcoumaran is the linkage of heterocycles, and is formed by β-5′ and αO-4′. During depolymerization, the α-O-4′ of the phenylcoumaran is readily hydrolyzed, resulting in the disruption of phenylcoumaran 182
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Fig. 6. Structural transformation of lignin during moderate ethanol/acid catalysis depolymerization.
structure. In addition, p-coumaric acid, which is typically connected to lignin by ester linkages, is dissolved owing to the breaking of ester linkages in the acid system. These hypotheses also additionally supply reasonable interpretations for the results of FT-IR, Py-GC/MS, 13C NMR, and HSQC NMR analyses. 3.3. Antioxidant activity of depolymerized lignin In order to explore the antioxidant activity of the depolymerized lignin, the total phenolics content, which is positively correlated to the antioxidant activity of lignin, was determined (An et al., 2017). As shown in Table 3, the depolymerized products at different reaction temperatures exhibited significant changes in the total phenolics content. The total phenolics contents of D-160, D-180 and D-200 were 145.9 ± 4.5, 149.9 ± 4.8 and 167.0 ± 3.6 mg GAE/g, respectively, and were visibly elevated compared to that of EIL (105.7 ± 4.6 mg GAE/g). Moreover, with the increase in S0, the total phenolics content further increased, which agreed with the decrease in molecular weight (Barapatre et al., 2015). Based on the above structural characterizations, the antioxidant
Fig. 7. DPPH free radical scavenging capacity of EIL and depolymerized products obtained at reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200). BHT was used as a positive control.
activities of the depolymerized products were further evaluated by a DPPH free radical scavenging assay. DPPH free radical scavenging capacities and IC50 values of all lignin samples are listed in Fig. 7 and Table 3, respectively. As shown in Table 3, the IC50 of D-160 (92.8 ± 3.6 μg/mL) was obviously reduced compared with the IC50 of EIL (168.1 ± 2.2 μg/mL), which implied that ethanol/acid catalysis resulted in the improvement of antioxidant activity. In addition, with the reaction temperature increasing up to 180 °C and 200 °C, the depolymerized products (D-180 and D-200) with lower molecular weight and higher total phenolics content exhibited lower IC50 values (83.9 ± 4.1 μg/mL and 66.0 ± 2.6 μg/mL, respectively). Although the depolymerized products (D-180 and D-200) possessed similar molecular weight, the DPPH free radical scavenging capacities of D-180 and D-200 were significantly different, possibly because D-200 has a lower polydispersity index and higher solubility (Dizhbite et al., 2004; Pan et al., 2006). Notably, the DPPH radical scavenging capacity of D-200 (IC50 = 66.0 ± 2.6 μg/mL) was close to that of the positive control
Table 3 Total phenolics content and DPPH free radical scavenging capacities (IC50 value) of EIL and depolymerized products obtained with reaction temperatures 160 °C (D-160), 180 °C (D-180) and 200 °C (D-200). Sample/condition
Total phenolics content/ (mg GAE/g)a*
IC50 (μg/mL)b*
EIL / 0 °C D-160/160 °C D-180/180 °C D-200/200 °C BHT
105.7 145.9 149.9 167.0 –
168.1 ± 2.2 92.8 ± 3.6 83.9 ± 4.1 66.0 ± 2.6 53.3 ± 4.5
± ± ± ±
4.6 4.5 4.8 3.6
a*: The total phenolics content of the sample was expressed as gallic acid equivalent (GAE). The calibration curve of gallic acid in DMSO (with six different concentrations from 4 μg/mL-20 μg/mL) was drawn in advance. b*: For a more intuitive display of the results, the IC50 values (the concentration required with 50% inhibition of radical formation) were further calculated based on the RSA with different sample concentrations. 183
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BHT (IC50 = 53.3 ± 4.5 μg/mL), indicating that the lignin sample after depolymerization has promising potential for the application as natural antioxidants. These results suggest that the cleavage of lignin ether linkages by ethanol/acid catalysis shortens the polymer chain and enhances the total phenolics content and antioxidant activity.
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